This proposal is divided into two sections, the first section describes experiments to investigate the nature of inverted repeated and other dispersed repeated sequences in mammalian genomes and a low molecular weight RNA found hydrogen-bonded to poly(A)-terminated RNAs from mammalian cells. The proposal is to investigate the positional relationship of these two ubiquitious sequences to protein coding sequences of mammalian DNA. The repeat sequences account for approximately 25% of the mammalian hnRNA by mass. They are also present in polyribosomal, poly(A)-terminated molecules and thus are of significant interest in the elucidation of mRNA formation in eukaryotes. Some evidence suggests that the repetitious sequences forming the inverted repeated DNA structures may be origins of DNA replication and we propose to investigate this possibility. In addition, because of the ubiquity of these sequences it is proposed that they might serve as preferential sites by which DNA introduced into eukaryotic cells might become integrated into the host cell'S DNA. This possibility will be investigated. The second part of the porposal describes experiments to investigate two enzymatic reactions known to play a role in mRNA production and maintainance in eukaryotes, RNA; RNA splicing and end addition to and shortening of poly(A) in mRNA molecules. We have developed an in vitro RNA:RNA splicing reaction that correctly breaks and rejoins RNA molecules to produce bona fide mRNA molecules from their precursor RNA molecules. The system will be purified further to obtain the active components. We have also isolated the cytoplasmic poly(A) polymerase from HeLa cells. This enzyme is absolutely dependent on divalent cations, has a mol. weight of 75,000 and apparantly is also a poly(A)-endonuclease, leaving a product of approx. 15-18 AMP residues with a 5' pAp and a 3' A-OH. We plan to further purify and characterize these two enzymatic activities as well as to determine whether the poly(A)-binding protein which also has a mol. weight of 75,000 is in fact the same protein. Considerable progress has already been made in each of these two projects.